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Also known as the 25-man roster, the active roster is the list of players who are available to play in a given game. During most of the regular season and all of the post-season, this roster is limited to 25 players. There is an exception for an extra player in doubleheaders, known at the 26th man rule. The roster is usually listed in the following order: pitchers, catchers, infielders and outfielders. There is no set number of players to be listed in each category, and players are not limited to playing the positions for which they are listed.

Most teams in the mid-2010s will carry a roster of 12 or 13 pitchers (5 starting pitchers and 7 or 8 relief pitchers), 2 catchers, 5 or 6 infielders (the four starters plus two or three substitutes) and 4 or 5 outfielders. This division has evolved over the years, and until the mid-1990s, it was common for teams to carry only 10 pitchers, leaving room for a third catcher and pinch-hitting or defensive specialists, for example.

A team's roster cannot be changed during a game, unless the game is suspended and resumed at a later date. Once in a while, a team will find itself in a roster crunch during a game, especially a high-scoring extra-inning game, when it runs out either of pitchers or position players and must use someone out of position. In order to minimize the risk of this happening, the Major Leagues have been granting teams added flexibility for roster management since the mid-2000s. For example, rules concerning the use of the disabled list were relaxed in the late 1980s. In 2003, a rule was introduced to allow a team to place a player on a compassionate list for a few days in order to replace him on the roster while he attends to family or personal obligations. A similar paternity list was added a few years later, for players whose wife was expecting to give birth momentarily. The 26th man rule, mentioned above, also stems from this concern.

In 2019, Major League Baseball decided to change the rule starting in 2020, by increasing the normal roster to 26 players until the periode of expanded rosters (see below).

Also known as the 40-man roster, the Major League roster lists all of the players who are signed to a Major League contract by a team. This will include its entire active roster, any players on the 15-day disabled list, plus some players in the team's farm system who are playing in the minor leagues on option. Players on the Major League roster can be recalled to the major league team at any time to fill an opening on the active roster, either due to injury, trade, or release of a player. If a team wants to add a player to its active roster who is not on its Major League roster, it must first clear a spot on that roster, either by releasing a player, revoking his option (which is called outrighting), or placing a player on the 60-day disabled list.

The Major League roster becomes important in the off-season, as players who are not retained on the roster can become free agents and be lost to the team. In addition, a team must return all players on the 60-day disabled list to the Major League roster, or risk losing them. But most importantly, a team must decide which of its minor league prospects deserve to be placed on the Major League roster; those who are left off and have been under contract for a certain number of seasons (three years for players signed after age 19, and four for those signed at age 19 or younger) are exposed to the Rule V Draft and can be claimed by any other team which has room left on its Major League roster. Future superstars such as Roberto Clemente and George Bell have been lost to their original teams because they had failed to place them on their Major League roster in the off-season.

Beginning on September 1st each season, teams could recall or "bring up" all of the players on their Major League roster and use them in games. This is known as the period of expanded rosters. In practice, most teams waited until their minor league affiliates' seasons were finished to call up players, and only called up a half dozen additional players or so, in order to evaluate what they could do against Major League competition. However, once in a while, some teams added a slew of players to its active roster, often because it needed a number of players with very specialized skills to help it during a pennant race, or to conduct a sort of advance spring training for the following season, while other teams declined the opportunity. This sometimes led to contests between teams with vastly different roster sizes, a problem which was increasingly pointed out during the 2010s.

In 2019, MLB decided to change the rule regarding expanded rosters starting with the 2020 season, in conjunction with expanding the regular roster by one player to 26. As of September 1st, teams will be formally required to add two and only two players to their roster, with the roster size remaining at 28 until the end of the season. This was done to ensure that any game would feature teams with an equal number of players, and that the nature of the game would not be changed by an overabundance of pitchers or other substitutes available to enter the game.

The 25-player roster limit had a long history behind it. It had been in place since the 1920 season, having previously been limited to 21 players, although it was also at 25 for periods before World War I. Originally, the limit was only in effect from May 15th to September 1st. It was raised in both the season's first and last month in order to allow teams to try out young players against major league competition. The early tryout period ended in 1968, but the September period remained in effect after that.

Even though the limit was at 25 players, not every team carried a full roster, and until the 1940s, it was relatively common for teams to leave a few unfilled spots and to use them to evaluate players just signed out of college. These would occasionally be used in games, but most often would just practice with the team and sit on the bench during games until assigned to a minor league team. Similarly, coaches and batting practice pitchers would sometimes be used in games, or amateur players would be given a contract for a few days to replace an injured regular. Since team owners were not fond of paying people to sit idly on the bench, and especially to fork over travel and meal money for these while on the road, teams tended to be very conservative when it came to rosters.

Things changed with the advent of bonus baby rules in the early 1950s. In order to discourage teams from giving large signing bonuses to amateur players, a rule was devised that these players had to be put on the team's active roster for a certain time - two years at first - and not be sent to the minor leagues until that probationary period had elapsed. This began to serve as a disincentive because teams had begun to see the value in having certain specialized players on their roster, such as pinch-hitters, defensive specialists and relief pitchers. Having to carry two or three bonus babies who were hardly ever used in games became a serious hindrance to managers.

The normal roster limit was 25 for most of the past decades with a few exceptions. In April 1990, because spring training had been shortened by a labor conflict, teams were allowed two additional roster spots until April 25th. This precedent was repeated after the settlement of the 1994 strike, when teams were allowed three additional roster spots at the start of the 1995 season until May 15. There are also special circumstances such as when teams begin their season early in Japan or elsewhere; they are allowed to have some extra players on their roster for those games. Conversely, Major League teams decided to play with 24-man rosters during the first half of the 1978 season (i.e. until July 1st) and during the entire season - except for the period of expanded rosters - from 1986 to 1989, as a cost-cutting measure in the face of escalating player salaries. The settlement of the 1990 strike made the 25-man roster a part of the basic collective bargaining agreement and it was not been touched until 2019, when MLB decided, in agreement with the Players Association, to increase it to 26 players starting in 2020.